The present invention relates generally to the preparation and storage of the enzyme peroxidase, and more particularly to a method for treating a solution containing peroxidase enzyme to increase its stability and shelf life.
Peroxidase enzymes are fairly ubiquitous, occurring in higher plants, yeasts, molds, bacteria and mammals. The enzyme peroxidase, especially horseradish peroxidase, has many uses. Of particular importance is its use in reagents employed in immunoassays. Horseradish peroxidase has also recently been found to be useful in a biocide formulation. In the latter use, the enzyme is relied on for its ability to catalyze the oxidation of phenolic materials, (see U.S. Pat. No. 4,370,199 and U.S. Pat. No. 4,478,683, assigned to the present assignee). Nevertheless, the major obstacle to the widespread use of peroxidase enzymes particularly for industrial applications is that their desired qualities ae not maintainable over any substantial period of time when stored in low concentration either in the dissolved or suspended states. Microbial contamination appears to be the major contributing factor for the instability of peroxidase enzymes, however, peroxidase activity may also decrease during, or as a result of the manner in which they are stored. It is known that peroxidases are not very stable for long term storage at high tempertures (e.g. above 25 degrees C.) especially in solution. As a result, samples of peroxidase have to be stored in a cold condition (e.g. 4 degrees C. or less) and preferably in the solid state.
U.S. Pat. No. 4,169,012 and No. 4,228,240 each describe methods for stabilizing peroxidase compositions by adding polyvalent ions of Groups III and IV of the Periodic Table to reagents containing peroxidase compositions. U.S. Pat. No. 4,252,896 discloses a method of stabilizing peroxidase in a serum protein based medium with the addition of a stabilizingly effective amount of 8-anilino-1-naphthlenesulfonic acid (ANS). According to this method, the amount of activity remaining after 43 days storage at 45 degrees C. is about 43%. Finally, U.S. Pat. No. 4,448,882 discloses a method of stabilizing a peroxidase composition in a serum protein with the addition of a stabilizing amount of 4-amino-antipyrine (AAT). According to this method about 58% activity remains after 28 days storage at 37 degrees C.
In contrast to the above prior art, the present invention relates to a method for the stabilization of a peroxidase containing solution without the use of additives or stabilizers. Moreover, the method of the present invention produces a peroxidase composition that can be stored substantially indefinitely at room temperature. As a result, the present invention differs from U.S. Pat. No. 4,169,012 and U.S. Pat. No. 4,228,240 which require the addition of a polyvalent ion and storage in the freeze dried state, and from U.S. Pat. No. 4,252,896 and U.S. Pat. No. 4,448,882 which require the addition of stabilizing agents and storage at an elevated temperature.